Urine samples, preparation

Berg et al. [126] have developed a method for the determination of opiates and cocaine in urine. Sample preparation was performed by adding ISTDs and 0.5 ml of... 

Another product of esterase cleavage is p-nitrophenol (PNP), which is generated in the human body by the degradation of parathion, parathion-methyl and parathion-ethyl. Some methods for the determination of PNP in urine are summarized in Table 9.5. PNP can, for example, be determined in the urine of occupationally exposed subjects by means of GC-ECD. Sample preparation involves acid hydrolysis, extraction with diethyl ether, derivatization with diazoethane and purification on silica gel columns. The LOD was 20 pg L with a recovery of 85-98%. This method has also been used in a study on selected pesticide residues and metabolites in urine from a survey of the US general population.In the context of another survey study, a more complicated technique to detect PNP in urine has recently been used by Hill et involving GC-MS-MS with positive chemical ionization after derivatization with l-chloro-3-iodopropane. The authors also used this method for the determination of the metabolite TCP and 10 other analytes in urine.Sample preparation involved hydrolysis with p-glucuronidase, several extraction steps using different solvents and purification by SPE (silica gel column). The LOD was 1 pg L with an inter-assay RSD... 

Trocewicz, J. (2004). Urine sample preparation of tricyclic antidepressants by means of a supported Equid membrane technique for high-performance liquid chromatographic analysis. J. Chromatogr. B, 801, 213-20. 

Urine Sample Preparation with Sulfuric Add SPE Eluent... 

One approach is to prepare a sample blank using urine known to be free of quinine. The fluorescent signal for the sample blank is subtracted from the urine sample s measured fluorescence. 

Immunoassays. Immunoassays (qv) maybe simply defined as analytical techniques that use antibodies or antibody-related reagents for selective deterrnination of sample components (94). These make up some of the most powerflil and widespread techniques used in clinical chemistry. The main advantages of immunoassays are high selectivity, low limits of detection, and adaptibiUty for use in detecting most compounds of clinical interest. Because of their high selectivity, immunoassays can often be used even for complex samples such as urine or blood, with Httle or no sample preparation. 

The methods I- 4 of sample preparation are classics. As a mle they give a high value of blank and some of them take a lot of time. Microwave sample preparation is perspective, more convenient and much more faster procedure than classical mineralization. There are some problems with the combination Cendall-Kolthoff s kinetic method and microwave sample preparation which discussed. The experimental data of different complex organic matrix are demonstrated (food products on fat, peptides, hydrocarbone matrix, urine etc). 

The quantification of kinins in human tissues or body fluids has been limited due to the inherent difficulties in accurately measuring the concentration of ephemeral peptides. Today HPLC-based and RIA/capture-ELA measurements are established to determine kinins in human plasma, liquor or mine. Serine protease inhibitors need to be added to prevent rapid degradation of the kinins in vitro during sample preparation. Kinins and their degradation products have been studied in various biological milieus such as plasma/ serum, urine, joint fluids, kidney, lung and skeletal muscle [2]. Under normal conditions, the concentration of kinins in these compartments is extremely low for... 

Several methods are available for the analysis of trichloroethylene in biological media. The method of choice depends on the nature of the sample matrix cost of analysis required precision, accuracy, and detection limit and turnaround time of the method. The main analytical method used to analyze for the presence of trichloroethylene and its metabolites, trichloroethanol and TCA, in biological samples is separation by gas chromatography (GC) combined with detection by mass spectrometry (MS) or electron capture detection (ECD). Trichloroethylene and/or its metabolites have been detected in exhaled air, blood, urine, breast milk, and tissues. Details on sample preparation, analytical method, and sensitivity and accuracy of selected methods are provided in Table 6-1. 

For pesticide residue immunoassays, matrices may include surface or groundwater, soil, sediment and plant or animal tissue or fluids. Aqueous samples may not require preparation prior to analysis, other than concentration. For other matrices, extractions or other cleanup steps are needed and these steps require the integration of the extracting solvent with the immunoassay. When solvent extraction is required, solvent effects on the assay are determined during assay optimization. Another option is to extract in the desired solvent, then conduct a solvent exchange into a more miscible solvent. Immunoassays perform best with water-miscible solvents when solvent concentrations are below 20%. Our experience has been that nearly every matrix requires a complete validation. Various soil types and even urine samples from different animals within a species may cause enough variation that validation in only a few samples is not sufficient. 

Because of the possibility that the herbicide alachlor could adulterate food if either poultry or livestock consumed contaminated materials, Lehotay and Miller evaluated three commercial immunoassays in milk and urine samples from a cow dosed with alachlor. They found that milk samples needed to be diluted with appropriate solvents (1 2, v/v) to eliminate the matrix effect. One assay kit (selected based on cost) was also evaluated for use with eggs and liver samples from chickens. Egg and liver samples were blended with acetonitrile, filtered, and diluted with water. Linear calibration curves prepared from fortified egg and liver samples were identical... 

Field control and field-fortified urine samples were prepared with urine from individuals with no known exposure to chlorpyrifos. Some control urine samples were left unfortified (controls), and some control urine samples were spiked with a "low" amount of 3,5,6-TCP, shaken, and stored on dry ice until analysis. Some control samples were also fortified with a "high" amount of 3,5,6-TCP. 

An alternative system proved to be both simpler and more user friendly (Unger et al., 2004 Machtejevas et al., 2006). Thus far we have used this configuration to analyze human plasma, sputum, urine, cerebrospinal fluid, and rat plasma. For each particular analysis we set up an analytical system based on a simple but specific strategy (Figure 9.5). The analysis concept is based on an online sample preparation and a two-dimensional LC system preseparating the majority of the matrix components from the analytes that are retained on a RAM-SCX column followed by a solvent switch and transfer of the trapped peptides. The SCX elution used five salt steps created by mixing 20 mM phosphate buffer (pH 2.5) (eluent Al) and 20 mM phosphate buffer with 1.5 M sodium chloride (eluent Bl) in the following proportions 85/15 70/30 65/45 45/55 0/100 with at the constant 0.1 mL/min flow rate. Desorption of the... 

Additionally, the inj ected matrix must also be miscible with the solvents used in the separations. For normal phase mode separations, all water must be removed from the injected matrix. Since many of the complex matrixes, such as plasma, urine, and other biological fluids contain a large amount of water, this requires more time consuming sample preparation. However, water can be injected into a polar organic or reverse phase mode separation. Even within the same mode, mobile phases that are very different can cause large disturbances in the baseline. Oda et al., (1991) solved this problem by inserting a dilution tube followed by a trap column in order to dilute the mobile phase used on the achiral column. Following the dilution tube, a trap column was used to reconcentrate the analyte of interest before the enantiomeric separation. 

Because plasma and urine are both aqueous matrixes, reverse-phase or polar organic mode enantiomeric separations are usually preferred as these approaches usually requires less elaborate sample preparation. Protein-, cyclodextrin-, and macrocyclic glycopeptide-based chiral stationary phases are the most commonly employed CSPs in the reverse phase mode. Also reverse phase and polar organic mode are more compatible mobile phases for mass spectrometers using electrospray ionization. Normal phase enantiomeric separations require more sample preparation (usually with at least one evaporation-to-dryness step). Therefore, normal phase CSPs are only used when a satisfactory enantiomeric separation cannot be obtained in reverse phase or polar organic mode. 

With the increased popularity of LC-MS, the problem of overlapping enantiomer peaks from other amino acids has largely been resolved. The mass spectrometer can act as an additional dimension of separation (based on mass to charge ratio). Thus, only amino acids having the same mass-to-charge ratio must be separated achirally (see Desai and Armstrong, 2004). This additional dimension of separation also has implications for the applications in the matrices discussed previously. With the ability of the mass spectrometer to discriminate on the basis of mass, this lessens the need for complete achiral separation. For example, an LC-MS method was recently developed to study the pharmacokinetics of theanine enantiomers in rat plasma and urine without an achiral separation before the enantiomeric separation (Desai et al., 2005). In such matrices, proteins must still be removed by appropriate sample preparation. 

A GC-MS method for determining the isotope composition of lead in blood and urine samples is based on preparation of Pb(CgH4F-p)4 using the corresponding Grignard reagent108. 

Kato K. et al., 2005. Determination of 16 phthalate metabolites in urine using automated sample preparation and onhne preconcentration/high-performance liquid chromatography/tandem mass spectrometry. Anal Chem 77 2985. 

Probably the most effective use of XRF and TXRF continues to be in the analysis of samples of biological origin. For instance, TXRF has been used without a significant amount of sample preparation to determine the metal cofactors in enzyme complexes [86]. The protein content in a number of enzymes has been deduced through a TXRF of the sulfur content of the component methionine and cysteine [87]. It was found that for enzymes with low molecular weights and minor amounts of buffer components that a reliable determination of sulfur was possible. In other works, TXRF was used to determine trace elements in serum and homogenized brain samples [88], selenium and other trace elements in serum and urine [89], lead in whole human blood [90], and the Zn/Cu ratio in serum as a means to aid cancer diagnosis [91]. 

Sample preparation is rather involved. A sample of urine or fecal matter is obtained and treated with calcium phosphate to precipitate the plutonium from solution. This mixture is then centrifuged, and the solids that separate are dissolved in 8 M nitric acid and heated to convert the plutonium to the +4 oxidation state. This nitric acid solution is passed through an anion exchange column, and the plutonium is eluted from the column with a hydrochloric-hydroiodic acid solution. The solution is evaporated to dryness, and the sample is redissolved in a sodium sulfate solution and electroplated onto a stainless steel planchette. The alpha particles emitted from this electroplated material are measured by the alpha spectroscopy system, and the quantity of radioactive plutonium ingested is calculated. Approximately 2000 samples per year are prepared for alpha spectroscopy analysis. The work is performed in a clean room environment like that described in Workplace Scene 1.2. 

The urine of people who are heavy smokers contains mutagenic chemicals, chemicals that cause mutations in biological cells. Bioanalytical laboratories can analyze urine samples for these chemicals, but the samples must be cleaned up first prior to extraction with methylene chloride. The procedure for this cleanup utilizes an open column chromatography. Columns several inches tall and about an inch wide are prepared by packing them with an adsorbing resin that has been treated with methyl alcohol. The urine samples are passed through these columns as part of the sample preparation scheme. 

Stephanie Marquardt, a technician in the bioanalytical laboratory at the MDS Pharma Services laboratory in Lincoln, Nebraska, prepares open columns for the preparation of urine samples for analysis for mutagens. 

Even when hydrolysis and epimerization can be avoided during sample preparation and handling, it is not possible to conclude definitively whether the compounds found in plasma and urine are true metabolites or simply degradation products. Indeed, chemical degradation can also occur within the body since urine and plasma contain a wide variety of potential catalysts, including metal ions, phosphate ions, proteins, and sugars (see Sect. 5.2.6). Whereas the existence of mammalian enzymes that act on penicillins and cephalosporins is considered possible [155], no such mammalian enzyme appears to have been identified to date. 

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